Development of finely tuned liposome nanoplatform for macrophage depletion.

BACKGROUND: Immunotherapy with clodronate-encapsulated liposomes, which induce macrophage depletion, has been studied extensively. However, previously reported liposomal formulation-based drugs (Clodrosome® and m-Clodrosome®) are limited by their inconsistent size and therapeutic efficacy. Thus, we aimed to achieve consistent therapeutic effects by effectively depleting macrophages with uniform-sized liposomes. RESULTS: We developed four types of click chemistry-based liposome nanoplatforms that were uniformly sized and encapsulated with clodronate, for effective macrophage depletion, followed by conjugation with Man-N3 and radiolabeling. Functionalization with Man-N3 improves the specific targeting of M2 macrophages, and radioisotope labeling enables in vivo imaging of the liposome nanoplatforms. The functionalized liposome nanoplatforms are stable under physiological conditions. The difference in the biodistribution of the four liposome nanoplatforms in vivo were recorded using positron emission tomography imaging. Among the four platforms, the clodronate-encapsulated mannosylated liposome effectively depleted M2 macrophages in the normal liver and tumor microenvironment ex vivo compared to that by Clodrosome® and m-Clodrosome®. CONCLUSION: The newly-developed liposome nanoplatform, with finely tuned size control, high in vivo stability, and excellent ex vivo M2 macrophage targeting and depletion effects, is a promising macrophage-depleting agent.

Selective Detection of Nitrogen-Containing Compound Gases.

N-containing gaseous compounds, such as trimethylamine (TMA), triethylamine (TEA), ammonia (NH3), nitrogen monoxide (NO), and nitrogen dioxide (NO2) exude irritating odors and are harmful to the human respiratory system at high concentrations. In this study, we investigated the sensing responses of five sensor materials-Al-doped ZnO (AZO) nanoparticles (NPs), Pt-loaded AZO NPs, a Pt-loaded WO3 (Pt-WO3) thin film, an Au-loaded WO3 (Au-WO3) thin film, and N-doped graphene-to the five aforementioned gases at a concentration of 10 parts per million (ppm). The ZnO- and WO3-based materials exhibited n-type semiconducting behavior, and their responses to tertiary amines were significantly higher than those of nitric oxides. The N-doped graphene exhibited p-type semiconducting behavior and responded only to nitric oxides. The Au- and Pt-WO3 thin films exhibited extremely high responses of approximately 100,000 for 10 ppm of triethylamine (TEA) and approximately -2700 for 10 ppm of NO2, respectively. These sensing responses are superior to those of previously reported sensors based on semiconducting metal oxides. On the basis of the sensing response results, we drew radar plots, which indicated that selective pattern recognition could be achieved by using the five sensing materials together. Thus, we demonstrated the possibility to distinguish each type of gas by applying the patterns to recognition techniques.

Thin PEGylated Carbon Nitrides: Water-Dispersible Organic Nanodots as Bioimaging Probes.

Fluorescent materials are being used for the optical/fluorescence imaging of living cells and animal models. As such, the development of heavy-metal-free, water-dispersible, and biocompatible imaging probes is still important. Carbon nitride (C3 N4 ) is used as a bioimaging probe due to its suitable optical properties, thus enhancing its biocompatibility and dispersibility in aqueous media is required. In this study, we incorporated short-chain polyethylene glycol (PEG) groups onto a carbon nitride network by the simple N-alkylation of hexaethylene glycolic mesylate with nucleophilic nitrogen atoms on oxidized carbon nitride (OCN). The PEGylated OCN (PEG-OCN) was well dispersed in water as nanodots with a lateral dimension of approximately 30 nm and a thickness of 0.5-1.2 nm and showed strong photoluminescence in the visible region. Cell-viability testing confirmed that these "heavy-metal-free" organic nanodots were highly biocompatible and noncytotoxic. In particular, the developed nanodots could provide clear confocal images of RAW 264.7 cells without weakening cell activity and displaying any aggregation in a range of concentrations (25-100 µg mL-1 ) with bright-green emission in the cytoplasm.

Sensing Properties of ZnO Nanoparticles for Detection of 2-Chloroethyl Ethyl Sulfide as a Mustard Simulant.

In this work, we present the fabrication and characterization of a 2-chloroethyl ethyl sulfide (2-CEES) gas sensor based on ZnO nanoparticles (NPs) synthesized by a hydrothermal method. We confirmed that synthesized ZnO NPs adopt a polycrystalline phase. Partially aggregated ZnO-NPs revealed spherical or ellipsoidal nanocrystalline particles in a size range of 30-50 nm, as observed by field-emission scanning electron microscopy (FE-SEM). The maximum response of the ZnO NPs was 15 at 1 ppm 2-CEES concentration, and a low detection limit of 0.4 ppm was observed at an optimal operating temperature of 250 °C. The lowest response time was 6 s in 20 ppm at 250 °C. The linearity response with correlation coefficient (R2) was 0.9887 at 2-CEE concentrations of 0.4-1 ppm at the operating temperature of 250 °C. The enhanced sensing performance and a decrease in the operating temperature were attributed to a high specific surface area and more active sites in the ZnO NPs after exposure to 2-CEES.

Complete genome analysis of a novel umbravirus-polerovirus combination isolated from Ixeridium dentatum.

Two novel viruses, isolated in Bonghwa, Republic of Korea, from an Ixeridium dentatum plant with yellowing mottle symptoms, have been provisionally named Ixeridium yellow mottle-associated virus 1 (IxYMaV-1) and Ixeridium yellow mottle-associated virus 2 (IxYMaV-2). IxYMaV-1 has a genome of 6,017 nucleotides sharing a 56.4% sequence identity with that of cucurbit aphid-borne yellows virus (genus Polerovirus). The IxYMaV-2 genome of 4,196 nucleotides has a sequence identity of less than 48.3% with e other species classified within the genus Umbravirus. Genome properties and phylogenetic analysis suggested that IxYMaV-1 and -2 are representative isolates of new species classifiable within the genus Polerovirus and Umbravirus, respectively.

Evaluation of [(89)Zr]-Oxalate as a PET Tracer in Inflammation, Tumor, and Rheumatoid Arthritis Models.

To obtain an additional pharmacological agent for the diagnosis of inflammation, we investigated the medical use of (89)Zr-oxalate as a positron emission tomography (PET) probe for the in vivo imaging of inflammation and compared its efficacy to that of 2-deoxy-2-[(18)F]fluoro-d-glucose ([(18)F]FDG) and sodium [(18)F]fluoride. (89)Zr-oxalate exhibited observable higher uptake in a macrophage cell line than in tumor cells. The inflammatory lesions and tumors were clearly visualized by PET imaging and autoradiography using (89)Zr-oxalate. Compared to [(18)F]FDG and sodium [(18)F]fluoride, (89)Zr-oxalate demonstrated a high selectivity index to the tumor at an early time point after injection and to inflammation at a delayed time point after injection (24 h). Through histological examination, large numbers of macrophages and neutrophils were observed in the tumor lesions with the highest (89)Zr-oxalate uptake. In a rheumatoid arthritis (RA) mouse model, (89)Zr-oxalate demonstrated a high level of accumulation in inflammatory lesions. (89)Zr-oxalate is a new strategic tool for tumor imaging and inflammatory processes.

The complete genomic sequence of a tentative new polerovirus identified in barley in South Korea.

The complete nucleotide sequence of a new barley polerovirus, tentatively named barley virus G (BVG), which was isolated in Gimje, South Korea, has been determined using an RNA sequencing technique combined with polymerase chain reaction methods. The viral genomic RNA of BVG is 5,620 nucleotides long and contains six typical open reading frames commonly observed in other poleroviruses. Sequence comparisons revealed that BVG is most closely related to maize yellow dwarf virus-RMV, with the highest amino acid identities being less than 90 % for all of the corresponding proteins. These results suggested that BVG is a member of a new species in the genus Polerovirus.

Accuracy of four different digital intraoral scanners: effects of the presence of orthodontic brackets and wire.

OBJECTIVE: The objective of this study was to compare the accuracy of four different digital intraoral scanners and the effects of buccal brackets and orthodontic wire. METHODS: For this study, three sets of models (Control model, BKT model with buccal bracket, and WBKT model with buccal bracket and orthodontic wire) were scanned using four different types of intraoral scanners: E4D dentist, iTero, Trios, and Zfx IntraScan. The mesiodistal width of the teeth, intercanine width, and intermolar width measured by four scanners were compared. Three-dimensional (3D) images of the brackets were taken using the four scanners. Data were analyzed with one-way ANOVA, independent t test, and post-hoc Tukey test at a significance level of P < 0.05. RESULTS: When comparing the 3D images with manual measurements using a traditional caliper, iTero and Trios showed the highest accuracy in horizontal measurements.iTero had the lowest values in Devmax-min of maxillary intermolar and intercanine widths (0.16 mm and 0.20 mm, respectively), whereas Trios had the lowest values in Devmax-min of mandibular intermolar and intercanine widths (0.36 mm and 0.14 mm, respectively). The horizontal variables were barely affected by the presence of buccal brackets and orthodontic wire. Comparison of 3D bracket images scanned by the four scanners showed differences in image distortion among the scanners. Bracket characteristics did not affect the 3D bracket images. CONCLUSIONS: The four intraoral scanners used in this study differed in accuracy. However, the results acquired by iTero and Trios were more reliable. Effects of buccal brackets and orthodontic wire on the 3D images taken by intraoral scanners were not clinically significant.

Oxidized carbon nitrides: water-dispersible, atomically thin carbon nitride-based nanodots and their performances as bioimaging probes.

Three-dimensional (3D) carbon nitride (C3 N4 )-based materials show excellent performance in a wide range of applications because of their suitable band structures. To realize the great promise of two-dimensional (2D) allotropes of various 3D materials, it is highly important to develop routes for the production of 2D C3 N4 materials, which are one-atom thick, in order to understand their intrinsic properties and identify their possible applications. In this work, water-dispersible, atomically thin, and small carbon nitride nanodots were produced using the chemical oxidation of graphitic C3 N4 . Various analyses, including X-ray diffraction, X-ray photoelectron, Fourier-transform infrared spectroscopy, and combustion-based elemental analysis, and thermogravimetric analysis, confirmed the production of 3D oxidized C3 N4 materials. The 2D C3 N4 nanodots were successfully exfoliated as individual single layers; their lateral dimension was several tens of nanometers. They showed strong photoluminescence in the visible region as well as excellent performances as cell-imaging probes in an in vitro study using confocal fluorescence microscopy.

Nucleotide sequence and genome organization of a new proposed crinivirus, tetterwort vein chlorosis virus.

The genome of tetterwort vein chlorosis virus (TVCV) from South Korea has been completely sequenced. Its genomic organization resembles those of other criniviruses, with several new features, indicating that TVCV is a member of a new species in the genus Crinivirus, family Closteroviridae. RNA1 contains 8467 nucleotides, with at least four opening reading frames (ORFs). ORF1a encodes a protein with predicted papain-like protease, methyltransferase, and helicase activities. ORF1b encodes a putative RNA-dependent RNA polymerase that is apparently expressed through a +1 ribosomal frameshift. RNA2 contains 8113 nucleotides encoding at least nine proteins, similar to most crinivirus RNA2s. The 3' untranslated regions of the bipartite RNA genome share 82.1% nucleotide sequence identity.

Nucleotide sequence and genome organization of atractylodes mottle virus, a new member of the genus Carlavirus.

The complete genome sequence of a member of a distinct species of the genus Carlavirus in the family Betaflexiviridae, tentatively named atractylodes mottle virus (AtrMoV), has been determined. Analysis of its genomic organization indicates that it has a single-stranded, positive-sense genomic RNA of 8866 nucleotides, excluding the poly(A) tail, and consists of six open reading frames typical of members of the genus Carlavirus. The individual open reading frames of AtrMoV show moderately low sequence similarity to those of other carlaviruses at the nucleotide and amino acid sequence levels. Pairwise comparison and phylogenetic analysis suggest that AtrMoV is most closely related to chrysanthemum virus B.

Complete genome sequence of a novel endornavirus isolated from hot pepper.

The complete genome of a putative new endornavirus infecting hot peppers (Capsicum annuum) was determined to be 14,729 nt in size, including 12 cytosines at the 3' end. The hot pepper-infecting virus has the highest nucleotide sequence similarity (94% query cover and 72% identity) to bell pepper endornavirus (BPEV) isolated from the cultivar Yolo Wonder in the USA (GenBank accession no. JN019858). The putative single, large open reading frame encodes a 4,884-amino-acid-long polyprotein that contains four putative functional domains: a viral methyltransferase, a viral RNA helicase, a glycosyltransferase, and an RNA-dependent RNA polymerase. A phylogenetic tree based on whole polyprotein sequences confirmed the close evolutionary relationship of the studied endornavirus to BPEV. The hot pepper-infecting virus also has a nick at nt position 975. Taken together, these results suggest that this virus belongs to a new species in the genus Endornavirus (family Endornaviridae), for which the name hot pepper endornavirus (HPEV) is proposed.

Genome sequence of a recombinant brassica yellows virus infecting Chinese cabbage.

RNA from a Chinese cabbage plant (Brassica campestris ssp. pekinensis) showing leaf malformation and mottling was labeled and hybridized to a DNA chip capable of detecting plant viruses and viroids. Probes specific for beet mild yellowing virus (BMYV) and beet western yellows virus (BWYV) yielded positive results, suggesting that the plant was infected by a polerovirus. Primers designed from the sequences of the positive probes were used to amplify and sequence one portion of the viral genome. This sequence showed a 90 % or greater identity to several poleroviruses, including BMYV, BWYV, beet chlorosis virus (BChV) and turnip yellows virus (TuYV). The complete genome sequence of the Chinese cabbage-infecting polerovirus consisted of 5,666 nt and was most closely related to brassica yellows virus (BrYV; 94 % identity). The virus was named BrYV-Cheongsong (BrYV-CS). However, ORF3, ORF4 and the 5' half of ORF5 of BrYV-CS were more closely related to those of TuYV, BWYV, BChV and BMYV than to those of BrYV. Interestingly, a recombination event (positions 3531-4819 in BrYV-CS) was detected when this sequence was aligned with those of BrYV and TuYV. This region showed the highest sequence identity to that of TuYV (94 % identity) and had greater than 93 % identity to those of BWYV, BChV and BMYV, but it shared only 81 % identity with that of BrYV. Taken together, the genomes of BrYV-CS and BrYV are closely related. However, the structural genes in the 3' half of the genome of BrYV-CS are more closely related to those of other poleroviruses.

The complete nucleotide sequence and genome organization of lychnis mottle virus.

The complete genomic sequence of lychnis mottle virus (LycMoV) from a Lychnis cognata plant was determined. LycMoV has a bipartite genome consisting of RNA1 (7,428 nt) and RNA2 (3,734 nt). Species in the family Secoviridae are demarcated based on their amino acid similarities in the protease-polymerase and coat protein. In LycMoV, these proteins share 90% and 63% sequence similarity, respectively, with the most closely related virus, strawberry latent ringspot virus, which is a member of the family Secoviridae but has not been assigned to a genus. Therefore, LycMoV is a tentative new virus of the family Secoviridae.

Complete genome sequence of a tentative new caulimovirus from the medicinal plant Atractylodes macrocephala.

A total of nine contigs related to caulimovirus-like sequences were detected using high-throughput paired-end RNA sequencing. An attempt to find the plant sample infected with this type of virus identified the medicinal plant Atractylodes macrocephala Koidzumi showing mild mottle symptoms. Subsequently, the complete DNA genome sequence of the Atractylodes virus was determined. The 8,105-nt genome of the virus was composed of six open reading frames and displayed the highest nucleotide sequence identity (70%) with soybean Putnam virus. Based upon the symptoms observed on the source plant, we propose to refer to this new member of the genus Caulimovirus as atractylodes mild mottle virus.

The complete genome sequences of two isolates of cnidium vein yellowing virus, a tentative new member of the family Secoviridae.

We determined the complete genome sequences of two isolates of cnidium vein yellowing virus (CnVYV-1 and -2) that co-infected all field samples collected from Cnidium officinale in Korea. Unlike CnVYV-2, however, CnVYV-1 was sap-transmissible to Nicotiana benthamiana. CnVYV-1 and -2 have bipartite genomes of 7,263 and 3,110 nucleotides and 7,278 and 3,112 nucleotides, respectively, excluding the poly(A) tails. Phylogenetic analysis of the CnVYV-1 and -2 sequences indicated close relationships to strawberry latent ringspot virus, an unassigned member of the family Secoviridae. CnVYV-1 and CnVYV-2 are closely related viruses that may represent a tentative new species of the family Secoviridae.

Genomic detection and characterization of a Korean isolate of Little cherry virus 1 sampled from a peach tree.

A peach tree (Prunus persica) showing yellowing and mild mottle symptoms was analyzed using high-throughput RNA sequencing to determine the causal agent. A total of nine contigs similar to Little cherry virus 1 (LChV-1) were produced, and all the contigs showed nucleotide sequence identity (lower than 83 %) and query coverage (higher than 73 %) with LChV-1. The symptomatic peach sample was confirmed to be infected with LChV-1-like virus as a result of reverse transcription-polymerase chain reaction using primers designed based on sequences of the contigs. Occurrence of diseases caused by LChV-1 in Prunus species has been reported. Complete 16,931-nt genome of the peach virus composed of eight open reading frames was determined, and conserved domains including viral methyltransferase, viral helicase 1, RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (HSP70h), HSP90h and closterovirus coat protein (CP) were identified. Phylogenetic trees based on amino acid sequence alignments between the peach virus and members in the family Closteroviridae showed that the virus was most similar to LChV-1. Pairwise comparisons based on amino acid sequence alignments of three genes (RdRp, HSP70h and CP) between the peach virus and LChV-1 isolates showed the highest amino acid sequence identities, with 84.32 % for RdRp, 85.48 % for HSP70h and 80.45 % for CP. These results indicate that this is the first report for the presence of LChV-1 in South Korea and may be one of the first reports of natural infection of peach by LChV-1. Although it is not clear if LChV-1 YD isolate was responsible for specific symptoms observed, detection and characterization of the peach tree-infecting LChV-1 in South Korea would be useful in terms of the epidemiology of LChV-1.

Complete genome sequence and construction of infectious full-length cDNA clones of tobacco ringspot Nepovirus, a viral pathogen causing bud blight in soybean.

Tobacco ringspot virus (TRSV, genus Nepovirus), causes severe diseases in soybean and tobacco plants. TRSV-induced bud blight disease significantly reduced both the yield and quality of soybeans. The function of the encoded viral gene product involved in TRSV infection was unclear due to the limitation of reverse genetics studies on the viral genome. Here, we represent the successful construction of infectious full-length cDNA clones of TRSV genome (RNA1 and RNA2). The cDNAs of TRSV RNA1 and RNA2 were cloned into the binary vector pPZP211 immediately downstream of a double cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase terminator. Seven days after agrobacterium-mediated co-inoculation of these two constructs, Nicotiana benthamiana plants developed a systemic infection with necrotic ringspot symptoms and weak stunting of the leaves, similar to that induced by natural TRSV. The systemic infection was confirmed by transmission electron microscopy and Western blot analysis. Simultaneously, soybean, tomato, and Arabidopsis ecotype Estland were mechanically inoculated with sap prepared from TRSV-agroinfiltrated N. benthamiana leaves, showing typical symptoms of bud blight, necrotic spots, and lethal systemic necrosis, respectively. The system developed herein will be an appealing way to determine TRSV viral gene functions and study host-TRSV interactions.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors.

Rice mitogen-activated protein kinase interactome analysis using the yeast two-hybrid system.

Mitogen-activated protein kinase (MAPK) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. Yet, our knowledge of the global physical MAPK interactome in plants remains largely fragmented. Here, we utilized the yeast two-hybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localization, and kinase assay experiments in the model crop rice (Oryza sativa) to systematically map what is to our knowledge the first plant MAPK-interacting proteins. We identified 80 nonredundant interacting protein pairs (74 nonredundant interactors) for rice MAPKs and elucidated the novel proteome-wide network of MAPK interactors. The established interactome contains four membrane-associated proteins, seven MAP2Ks (for MAPK kinase), four MAPKs, and 59 putative substrates, including 18 transcription factors. Several interactors were also validated by experimental approaches (in vivo and in vitro) and literature survey. Our results highlight the importance of OsMPK1, an ortholog of tobacco (Nicotiana benthamiana) salicyclic acid-induced protein kinase and Arabidopsis (Arabidopsis thaliana) AtMPK6, among the rice MAPKs, as it alone interacts with 41 unique proteins (51.2% of the mapped MAPK interaction network). Additionally, Gene Ontology classification of interacting proteins into 34 functional categories suggested MAPK participation in diverse physiological functions. Together, the results obtained essentially enhance our knowledge of the MAPK-interacting protein network and provide a valuable research resource for developing a nearly complete map of the rice MAPK interactome.